CA1330036C

CA1330036C - Inhibition of arterial thrombotic occlusion or thromboembolism

Info

Publication number: CA1330036C
Application number: CA000583138A
Authority: CA
Inventors: John H. Griffin; Andras Gruber; Stephen R. Hanson; Lawrence A. Harker
Original assignee: Scripps Clinic and Research Foundation
Current assignee: Scripps Research Institute
Priority date: 1987-11-17
Filing date: 1988-11-15
Publication date: 1994-06-07
Anticipated expiration: 2011-06-07
Also published as: FI104790B; DK640088A; EP0318201B1; NO301747B1; DK640088D0; ATE135234T1; DK175704B1; GR3020036T3; FI885331A0; FI885331A; JPH01238536A; NO885109L; EP0318201A2; US5350578A; ES2086301T3; DE3855096T2; DE3855096D1; EP0318201A3; NO885109D0; US5084274A

Abstract

ABSTRACT

The method of preventing arterial thrombotic occlusion or thromboembolism by administering plasma-derived or recombinant produced activated protein C alone or in combination with a thrombolytic agent or combina-tions of thrombolytic agents.

Description

3 . THROMBOEMBOLISM

4 ~ .
S 1l Backqround of the Invention 7 I This invention relates to the inhibition of arterial 8 ~ thrombotic occlusion or thromboembolism by plasma-derived or 9 I recombinant produced activated protein C (APC) alone or in ~ combination with a thrombolytic agent.
11 ~ Many of the surgical procedures carry the risk of 12 ~' venous and arterial thrombosis and thromboembolism.
13 1 Application of current anti-platelet or fibrinolytic drugs in 14 1 intraoperative or postoperative cases could lead to serious lS ~ bleeding complications. Thus, the use of these agents requires 16 1'l extra precaution. Even in diseases complicated with arterial 17 ~ thrombosis, the use of antithrombotic and/or thrombolytic 18 ~I therapy has undesired side effects, such as bleeding or 19 1 reocclusion during thrombolytic treatment in myocardial infarction, bleeding or thrombosis following surgery, and 21 thrombosis following surgery that employs grafts or other 22 cardiovascular prosthetic devices.
23 Therefore, there is a need for an antithrombotic 24 therapy which would be anticoagulant, anti-platelet and fibrinolytic at the same time without the hazards of 26 ll hemorrhage. APC is unique among the physiologic anticoagulants 27 ,, since it inhibits coagulation and stimulates fibrinolysis. APC
28 ~, inhibits the thrombin mediated activation of platelets as well 29 1 as the formation of fibrin, and thus, the formation of arterial 30 , thrombus built up mostly by platelets and fibrin. The use of `` 1 330036 ..

, `
I APC reduces the dose of tissue-type plasminogen activator 2 ll (t-PA) or other thrombolytic agents by its actions. Thus, APC
3 ! provides safer thrombolysis with less risk of bleeding and less 4 risk of reocclusion.
APC is a potent anticoagulant enzyme in vitro and in 6 vivo. APC inhibits the blood coagulation pathways and the 7 formation of thrombin by proteolytic cleavage of F.Va and F.VIIIa, and also enhances fibrinolysis (Seegers et al., 9 Thrombosis Res., 1, 443-460 (1972); Kisiel, J. Clin. Invest., 64, 761-769 (1979); Marlar & Griffin, J. Clin. Invest., ~ 66, 1186-1189 (1980); Marlar et al., Blood, 59, 1067-1072 12 , (1982); Clouse & Comp, New Enql. J. Med., 314, 1298-1304 13 ~, (1986)). APC is generated from its circulating precursor, 14 l' namely from the vitamin K dependent protein C (PC), upon 15 1! activation by immobilized thrombin on the endothelium of blood 16 1I vessels (Mammen et al., Thromb. Diath. Haemorrh., 5, 218-249 17 ll (1960); Stenflo, J. Biol. Chem., 251, 355-363 (1976); Esmon &
~i --18 ¦¦ Owen, Proc. Natl. Acad. Sci. USA, 78, 2249-2252 (1981)). APC, l9 through the protein C pathway, serv~s as the enzyme central to .0 the negative feedback regulation of coagulation. Inherited 21 deficiency in PC is associated with venous thromboembolic 22 diseases (Griffin et al., J. Clin. Invest., 68, 1370-1373 23 (1981); Bertina et al., Thromb. Haemost., 48, 1-5 (1982);

24 Griffin, Seminars in Thrombosis and Hemostasis, 10, 162-166 (1984); Marciniak et al., Blood, 65, 15-20 (1985)), but 26 ~1 inherited protein C deficiency is not significantly associated 27 ! with arterial thrombosis (Coller et al., Arteriosclerosis, 7, 28 1~ 456-462 ~1987). Infusion of APC decreases blood coagulability 29 1~ in various animal models and prevents the coagulopathic and lethal effects in E.coli infusion in baboons (Comp & Esmon, J.

``- 1 330036 Clin. Invest., 68, 1221-1228 (1981); Comp et al., J.
Clin. Invest., 70, 127-134 (1982); Colucci et al., J.
Clin. Invest., 7~, 200-20~ (1984); Taylor et al., J.
Clin. Invest., 79, 918-925 (1987); ~urdick & Schaub, Thrombosis Res., 45, 413-419 (1987)). Infusion of a thrombolytic agent like t-PA into humans results in effective thrombolysis in acute myocardial infarction (AMI) Yusuf et a]., European Heart Journal, 6, 556-585 (1985); European Cooperative Study Group, Lancet., 842-847 (1985)).
SummarY of the Invention ~ The present invention comprises the use of plasma-derived or recombinant produced activated pro-tein C alone or in combination with a thrombolytic agent such as tissue plasminogen activator or its analogs, urokinase or its analogs, prourokinase or its analogs, streptokinase or its analogs, an acylated form of plasminogen or plasmin or their analogs and acylated streptokinase plasminogen complex for inhibiting acute arterial thrombotic occlusion, throm-boembolism, or stenosis in coronary, cerebral or peripheral arteries or in vascular grafts.

The present invention also relates to a pharma-ceutical composition for inhibiting acute arterial thrombotic occlusion, thromboembolism, or stenosis in coronary, cerebral or peripheral arteries or in vascu-lar grafts the composition comprising an effective amount of activated protein C in a pharmaceutically acceptable carrier.
rief DescriPtion of the Drawinqs A more complete appreciation of the present invention and many of the attendant advantages thereof will be readily obtained as the invention becomes bet-ter understood by reference to the following detaileddescription in connection : 1 33003~
I
,, ` I
1 with the accompanying drawings, wherein all figures refer to 2 the effects of infusion of APC alone or APC and a thrombolytic 3 ~ agent into baboons used for an arterial thrombosis model:
4 ¦ Figures la thru 3a show the effect of infusions of low 5 ¦ dose APC on the clotting of blood measured using activated 6 partial thromboplastin time assays (APTTs).

7 Figures 4a and 5a show the effect of infusions of high 8 dose APC on APTTs.

9 I Figures 6a and 7a show the effect of infusions of a 10 ~ combination of APC and t-PA on APTTs.
~ Figures lb thru 5b show the effect of APC and Figures 12 1 6b and 7b of APC plus t-PA on blood flow and bleeding time.
13 Figures lc thru 5c show the effect of APC infusion or 14 1l APC plus t-PA infusion (Figures 6c and 7c) on platelet 15 1l deposition (i.e. thrombus formation) in a Dacron graft from 16 1l analyses of radioimaging data of radiolabeled platelets.
17 ~¦ Figure 8 shows the effect of t-PA infusion on platelet 18 ¦, deposition (i.e. thrombus formation) in a Dacron graft from 19 I analyses of radioimaging data of radiolabeled platelets.
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21 Detailed DescriPtion of the Invention 23 The Thrombosis Model 24 An arterial thrombosis model has been tested and characterized in previous experiments (Hanson & Harker, 26 J. Clin. Invest., 75, 1591-1599 (1985); Hanson ~ Harker, 27 ~ Thromb. Haemostas., 53, 423-427 (1985); Hanson et al., 2~ ¦, Arteriosclerosis, S, 595-603 ~1985)). This model is useful in 29 1l judging the effect of drugs on arterial thrombus formation.
3~ ~ Male baboons weighing 10-12 kg were dewormed and observed for I

!i I

more than 40 days at our animal facility prior to the experiments. Permanent arteriovenous shunts were prepared between the femoral artery and vein using 3 rnm i.d. Silastic tubing. Subsequent interposition of 5 cm long, 9 mm i.d.
Dacron vascular grafts served as thrombogenic surface, inducing continuous platelet-fibrin thrombus formation until progressive occlusion of the graft at 70 ~ 20 min. Experimental results using recombinant produced APC in the baboon in this arterial thrombosis model in baboons were obtained that indicate similar results to the plasma-derived APC.

Administration of APC and t-PA
All baboons underwent at least one control experiment prior to APC administration. The APC was given by injecting one-fourth to one-third of the total PC dose as a bolus and the remaining three-fourths to two-thirds of the dose as continuous infusion for one hour. The interposition of the graft and bolus injection were made at time-0 and APC was administered at the proximal site of the Silastic shunt. The experiments were '0 divided into three groups; the "low dose" APC group received 2.0-3.4 mg ~total), the "high dose~ APC group received 11 mg (total), and the combination group received 2.1 mg APC plus 1.3 mg t-PA (total).

Preparation of APC
Human plasma prothrombin complex concentrate (Immuno AG, Vienna) was the source of protein C. A monoclonal antibody directed against the light chain of PC ~designated C3) was prepared and coupled to CNBromide activated Sepharose*9B
(Pharmacia, Uppsala, Sweden), and the prothrombin complex * Trade mark .. ~

" 1 330036 concentrate diluted in a hufer (0.~2 M~l Tris, 0.002 M/l EDTA;
O.002 M/l Benzamidine; 0.1 M~l NaCl, 0~075 mM/l pAPMSF, 0.02~
Na-azide, 0.02% Tween 20, pH 7.4 was applied on the column.
The PC was eluted with ~ M thiocyanate and dialyzed against Tris-buffered saline (0.01 M/l Tris; 0.14 M/l NaCl, pH 7.9).
Purified PC was activated using thrombin-Sepharose beads as descrihed in Marlar et al., slood, 59, 1067-1072 (1982).
The purified APC appeared on SDS-PAGE as two bands and no significant contamination (> 5%) by other proteins was detected. In some preparations, the trace amounts of thrombin veri~ied by clotting assay were separated by using either Bio-Rex 70 absorption or Fast Protein Liquid Chromatography (FPLC) chromatography on a Mono-Q column (Pharmacia, Uppsala, Sweden. The activity of the purified APC was measured in activated partial thromboplastin time (APTT) clotting assays (Marlar et al., Blood, 59, 1057-1072 (1982)) that measured anticoagulant activity and in chromogenic substrate assays, and results were compared to the activity of a previausly purified APC preparation and to the activity of APC generated by ~0 addition of the PC activator, Protac*(American Diagnostica, Greenwich, CT), to normal human plasma.

Amidolytic and Anticoaqulant AssaYs of Purified APC
~arious amounts (0.5 to 5 microliters) of APC solution or of reference purified APC solution (0.5 mg/ml) were added to 210 microliters of buffer containing 0.01 M Tris-HCl, 0.14 M NaCl, 1% ovalumbin, 0.02% sodium azide, 0.05% Tween*80, pH 8.0 and the samples were placed in microtiter plate wells (Corning*, Dynatech*-Immulon, or Costar~. After addition of 20 microliters of chromogenic substrate, S-2401* (4.6 mM), the * rrrade mark chanqe in absorbance of the sample was read using an ELISA
plate reader at room temperature. The amidolytic activity of the APC was determined by comparing the observed values to the reference values. The anticoagulant activity was determined using the APTT assay. In this assay, dilutions of normal human plasma (nhp) were made using protein C deficient plasma (pcdp) as diluent. 10~ microliters of these mixtures was mixed with ; 100 microliters of Protac reagent (American Diagnostica) and 100 microliters of APTT reagent (General Diagnostics). After 5 min incubation, 100 microliters of CaCl2 (50 mM~ was added and the clotting time determined. Since nhp contains 4.0 micrograms/ml of protein C, the activity of unknown APC
solutions was determined by comparison to the standard curves obtained for dilutions of nhp. In some experiments, standard curves for the amidolytic activity of APC were made using - dilutions of nhp activated by Protac. In some experiments, S-2366*was used in place of S-2401. The specific activity of all preparations of APC was in good agreement with values for APC based on protein content determined by absorbance at 280 nm '0 using an extinction coefficient of 1.4 per cm per mg~ml.
The doses of APC described here in the baboon experiments indicate the functional activity of the APC
preparations in comparison to normal human plasma and purified APC as standards. The purified APC preparations showed an anticoagulant effect on human and baboon plasmas using the APTT
assay, and cleaved chromogenic oligo peptide-paranitroanilide substrates, S-2365 and S-2901 ~Kabi, Stockholm, Sweden) in a concentration dependent manner. t-PA from a melanoma cell line was kindly provided by Dr. Desire Collen (Leuven, Belgium).
3~
* Trade mark , l I Studies for Establishing the Antithrombotic 2 ` _ Properties of APC in Arterial Thombosis 3 1~ Blood Flow in the Shunt 4 ¦ The blood flow was measured using a Doppler flow meter I fitted around the distal segment of the Silastic tubing. The 6 values were given in ml/min and were in the range of 100-200 7 ml/min (equal to 13.3 - 26.5 cm/sec velocity) providing 8 arterial flow conditions. The flow values were recorded at 9 regular intervals throughout the experiments. The method was 10 1l described in Hanson & Harker et al., Arteriosclerosis, ~ 5, 595-603 (1985).

3 ~ Platelet DePosition in the Dacron Graft 1' 14 ~, The deposition of " 'In-labeled platelets was 15 I detected by scintillation camera images of the graft. The 16 ~I platelet labeling methods and data analysis were the same as 1 7 ¦¦ described in Hanson & Harker, Thromb. Haemostas., 53, 423-427 18 1~ (1985) with the only modification that the equation was l9 i simplified for the number of platelets in the graft as follows:
~0 cPm-qraft X platelet count/ml 21 cmp/ml of whole blood 23 The duration of imaging also was extended to two hours from the 24 time (tS0) of initiating the graft and the APC bolus in the APC
experiments. Since platelet accumulation usually reached a 26 1l plateau in control experiments within one hour, this one hour 27 ll period was not exceeded with imaging the inhibition of platelet 28 ¦i deposition. Inhibition of platelet deposition in treated 29 1 animals was expressed as % of total number of platelets 30 ' deposited in the control experiments at 30 and 60 minutes .

I!

1 33~03~

timepoints. The equations for the calculation of inhibition are shown in Table I. Since platelet deposition depends on 3 1 platelet count (Harker & Hanson, Thromb. Haemostas., 53, 4 ¦ 423-427 (1985), corr-oction of inhibition of platelet deposition was made using the equation given in Table 1.

7 Bleedinq Times 8 Standardized template bleeding times were performed on 9 the shaved volar surface of the forearm before and during the 10 I experiment as described in Malpass et al., Blood, 57, 736-740 ll 1 (1981), with two incisions, 5 mm long, 1 mm deep each, at 12 ll 40 mm Hg inflation of the sphygmomanometer.
13 1l 14 li Tests for Anticoagulant Effects ll and for In Vivo Plasma Levels of APC

16 ' Anticoagulant effect of APC infusion was measured by Il performing APTT assays at regular intervals from arterial blood ¦ll drawn into sodium citrate. The test was done within 5 to 10 ~I minutes from sampling in order to minimalize the in vitro inhibition of APC by plasma protein C inhibitor(s). To 21 determine the circulating APC levels a chromogenic amidolytic 22 assay was developed. ELISA microtiter plates (Dynatech-Immunlon 23 or Costar) were coated with the anti-protein C monoclonal 24 antibody, C3, that does not influence the amidolytic activity 25 of the enzym~ significantly. Blood was drawn into 3.8%
26 citrate, 4.6% benzamidine solution (9:1), and the plasma 27 ¦ obtained after immediate centrifugation was kept on -80C until 28 ,' studie~. 10 microliters of this plasma sample was diluted to 29 1' 160-200 microliters with a Tris-buffered saline containing 1%
30 'Il BSA as carrier and a 0.36% benzamidine as enzyme inhibitor and 1:
g 1 ; was incubated for one hour at 37C in the antibody coated 2 1i wells. The solution was removed and the wells washed to remove 3 '~ unbound constituents and the benzamidine. Then a chromogenic 4 substrate, either S-2366 or S-2401, was added and the rate of cleavage of the substrate was measured spectrophotometrically.
6 Using standard APC dilutions, the APC concentration in the 7 plasma samples was calculated from the calibration curve.

9 Results Indicating the APC is Antithrombotic Under Arterial Flow Conditions ~ Figs. la thru 3a show the APTT prolongation in the 12 ! "low dose" experiments (open circles). 2.0 - 3.4 mg total dose 13 1, of human APC approximately doubled the APTT values on an 14 ! average. After termination of the APC infusion (vertical I arrow), the APTT values progressively decreased and the 16 ~ measured level of APC as based on amidolytic activity decreased 17 I (solid line) suggesting a circulation half life of 12-16 18 , minutes. Figs. 4a and 5a show the effect of ~high dose~ APC on 19 APTTs. Administration of 11 mg APC resulted in a 3-4 times prolongation in APTT in both experiments. The pattern of APTT
21 changes and the measured level of APC after termination of the 22 infusion indicate similar half-life values of APC
23 (approximately 12 min) for these higher dose of APC.
24 Combination of APC (2.1 mg) and t-PA (1.3 mg) had the same effect on APTTs as the low dose APC had alone (Figs. 6a and 2S 7a~. In none of the experiments was the APTT decreased to the 27 ¦, starting value (at 0 min) at the end of the observation (at 120 28 ~ min).
29 l, The same figures (Figs. la thru 7a) demonstrate the 30 1ll changes in the APC levels measured by the chromogenic assay I ~1 '........................................................... I
!
1 I described above (closed circles). An overall range of 0.38 to 2 ll o . 70 micrograms/ml of APC plasma concentration in the "low 3 Ij dose" experiments (Figs. la thru 3a), 0.94 to l.64 4 ¦¦ micrograms/ml in the "high dose" experiments (Figs. 4a and 5a) 5 !~ and 0.30 to 0.90 micrograms/ml in the combination experiments 6 1¦ ~Figs. 6a and 7a) were measured. The circulation half life of 7 ¦ APC in the baboons determined in amidolytic activity assays was 8 similar to that determined from the APTT determinations, 9 j' regardless of the total dose of the APC enzyme. The in vivo 10 ~ circulating APC levels did not entirely return to the starting values within two hours. The five fold difference in APC doses 12 ~l used in high dose compared to low dose experiments did not 13 !i result in five-fold higher circulating APC levels, suggesting 14 ll an efficient clearance or temporary storage mechanism or 15 ll receptor mediated regulation for APC.
16 I In six of the seven experiments the bleeding times 17 I remained in the normal range (Figs. lb and 3b thru 7b vertical 18 I segments) with a slight average elevation. In one of the "low 19 ¦~ dose~ experiments the bleeding times were prolonged and _0 abnormal (Fig. 2b), both before and during and after the 21 experiment. Since the bleeding time was abnormally long in 22 this animal before the APC infusion, the long bleeding time 23 observed dùring AP~ infusion was not due to APC. These results 24 suggest that circulating APC at the doses employed does not significantly alter the hemostatic platelet function measured 26 l, by standardized bleeding time techniques. There were no 27 j, suffusions, hematomas or rebleeding observed at tissue injury 28 `, sites (i.e. sewing cuffs) typical of higher doses of t-PA.
29 1I The blood flow was maintained undiminished in the j experiments during APC administration (Figs. lb thru 7b, solid I! ~

. . I
1 circles) in contrast to control experiments for the same 2 animals where occlusion regularly occurred. Six of the seven 3 grafts remained open throughout the whole observation period 4 ¦¦ with good arterial flow. In one of the APC-t-PA experiments 5 ~¦ (Fig. 6b) the Dacron graft failed at 115 minutes. In four of 6 ¦I the nine control studies the grafts occluded within one hour, 7 ¦¦ and these grafts fail in ~0 + 20 minutes when there is no effective antithrombotic agent in the circulation. The blood 9 ll flow changes in two control experiments are demonstrated in lO ! Fig. 3b and Fig. 7b. Fig. 3b shows the flow rates of the 11 second control, when the graft remained open up to 1 hr (the 12 ,~ first graft became occluded). In Fig. 7b rapid progressive 13 ~ occlusion of the control graft can be observed. These data 14 l, showed that APC alone or in combination with t-PA is 15 ll antithrombotic under arterial flow conditions. The high dose 16 il APC experiments showed long lasting antithrombotic effects 17 ¦I since the flow did not change significantly throughout two 18 1l hours.

19 il Figs. lc thru 7c show the results of analyses of ~ ¦ radioimaging data. The values express the total platelets 21 deposited in the Dacron graft for a maximum of one hour in the 22 control and two hours in the APC experiments. Solid circles 23 ( ) show the number of platelets deposited in arterial 24 thrombus formation when APC was infused while open circles I (----) show the results for control experiments done in the 26 l, same animals. The typical sigmoid curve can be seen in the 27 controls as described in Hanson ~ Harker, Thromb. Haemostas., 28 , 423-427 ~1985). The deposition of platelets in the graft was 29 ~ significantly inhibited in every APC or APC-t-PA experiment, compared to controls. The degree of inhibition was given in , 'I

i ,, I

1 ~ percentage in Table I. The corrected values for inhibition of 2 ~ platelet deposition (I2, Table l) were 34%, 52% and 42% in 3 j the "low dose" experiments at 30 min, respectively (mean:
4 43%). (Figs. lc thru 3c). In the "high dose" experiments the values were 74% and 64% at 30 min (mean: 69%) and 72% and 83%
6 at 60 min (mean: 78%) (Figs. 4c and 5c) and the APC-t-PA
7 experiments 53% and 36% at 30 min (mean: 45%) (Figs. 6c and 8 7c). A long-lasting inhibition of platelet deposition was seen 9 in the high dose studies after terminating APC infusion.
Fig. 8 shows the results of some previous studies with t-PA in ll j, the same experimental model. l mg of t-PA (O.l mg/kg/hour) 12 1 infusion had an intermediate antiplatelet effect in these 13 experiments.
14 ¦ our results provide evidence that human APC inhibits lS ¦ arterial thrombus information in a dose dependent manner and 16 I that combination of APC with t-PA inhibits arterial thrombus 17 ¦ formation. This shows that APC infusion could reduce the 18 antithrombotic dose of t-PA. These effects of APC are achieved 19 without a significant prolongation of the bleeding time and 0 without risks of bleeding.

22 Advantaqe 23 Human APC infusion alone or in combination with a 24 thrombolytic agent (t-PA) can be used as a therapeutic agent in humans with arterial thrombosis. The results of the studies 26 provide evidence that APC is a very potent antithrombotic agent 27 ¦¦ under arterial flow conditions at plasma levels of 0.24 to 1.6 28 l! micrograms/ml.
29 l! From the experiments it can be concluded that APC

30 ¦ alone or AP~ in combination with a thromobolytic agent is a , 1 1 markedly effective antithrombotic agent for complex thrombus 2 formation under arterial flow conditions.
3 APC alone or a combination of APC with a thrombolytic 4 agent such as t-PA potently inhibits the participation of both platelets and fibrin formation in acute arterial thrombosis of 6 a complex type, a process that is unresponsive to heparin or 7 currently available antiplatelet drugs when used alone or in 8 combination. Since the APC is a physiologic material, its 9 administration has potent antithrombotic effects without causing significant impairment of primary hemostasis (as 11 measured in bleeding time tests) or evident toxicity.

13 Application 14 Therapy using APC alone or APC in combination with a thrombolytic agent is useful for vascular disorders involving 16 arterial thrombosis.
17 Some examples of arterial thrombosis where APC alone 18 or in combination with a thrombolytic agent is useful include 19 the following clinical settings.
1. Acute arterial thrombotic occlusion including 21 coronary, cerebral, renal, mesenterial, pulmonary or 22 peripheral arteries.
23 2. Acute thrombotic occlusion or restenosis after 24 angioplasty.
3. Reocclusion or restenosis after thrombolytic 26 therapy. Thrombolytic agents such as t-PA salvage ischemic 27 tissue when used within hours of acute heart attack or stroke 28 by re-establishing blood flow in the occluded artery. At 29 present, between one-four and one-third of patients who have successful thrombolytic reperfusion of occluded coronary 31 arteries subse~uently undergo reocclusion after discontinuing 1 , t-PA infusion. This complication occurs despite full-dose 2 1; heparin therapy. APC will have greater efficacy than heparin 3 in preventing reocclusion.
4 1 4. Small and large caliber vascular graft occlusion. Vascular grafts of small caliber, i.e., 3-/mm 6 diameter, have a high frequency of thrombotic occlusion. APC
7 ¦ alone or in combination with a thrombolytic agent is useful to 8 j prevent occlusion.
9 ' 5. Hemodialysis. The prosthetic surfaces and flow 10 1~ design of all hemodialyzers are thrombogenic. Currently ~ heparin is infused during dialysis. However, heparin is only 12 ll partially effective, thereby limiting the reuse of dialyzers.
13 , Also, heparin has a number of troublesome side effects and 14 ll complications.
15 1l 6. Cardiopulmonary bypass surgery. To prevent 16 ¦~ thrombus formation in the oxygenator and pump apparatus, 17 ¦I heparin is currently used. However, it fails to inhibit 18 1~ platelet activation and the resultant transient platelet 19 I dysfunction which predisposes to bleeding problems post-operatively.
21 7. Left ventricular cardiac assist device. This 22 prosthetic pump is highly thrombogenic and results in life 23 threatening thromboembolic events -- complications that are 24 only partially reduced by convential anticoagulants (heparin or coumarin drugs).
26 ! 8. Total artificial heart and left ventricular 27 I assist devices.
28 11 9. Other arterial thrombosis. APC is useful for 29 'i arterial thrombosis or thromboembolism where current 30 1l therapeutic measures are either contraindicated or not 1, - ;5 -t 330036 .~ I

1 , effective. For example, APC is useful for the treatment of 2 , acute pre-or postcapillary occlusion, including 3 ',, transplantations, retina thrombosis, or microthrombotic 4 necrosis of any organ complicating infections, tumors, or S coumarin treatment.
6 In summary, human APC, a naturally occurring 7 physiologic antithrombotic human protein, is superior to other available antithrombotic drugs in terms of bleeding tendency 9 (heparin, thrombolytic drugs, antiplatelet drugs), toxicity 10 ll (some antiplatelet drugs), antigenicity (streptokinase), clearance rate (heparin, antiplatelet drugs, teratogenicity 12 1l (coumarin derivatives), general side effects ~antiplatelet 13 ¦I drugs), lack of immediate efficacy (antiplatelet drugs, 14 ll coumarin derivatives), allergic reactions (antiplatelet drugs, 15 1I streptokinase, heparin), and hypotensive effect (prostacyclin).
16 1, APC combined with t-PA or another thrombolytic agent 17 1l improves the antithrombotic effect of a thrombolytic agent 18 1 alone. Thus, APC therapy will reduce the doses of t-PA or 19 I other thrombolytic agents required for therapeutic treatment of thrombosis, thereby avoiding the complications of high doses of 21 thrombolytic agents.
22The above description provides details o~ the manner ~3 in which the embodiments of the present invention may be made 24 and used. This description, while exemplary of the present invention, is not to be construed as specifically limiting the 26 1l invention and such variations which would be within the purview 27 11 of one skilled in the art are to be considered to fall within 28 ,I the scope of this invention.

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1 I TABlE 1 2 ~1INHIBITION OF GRAFT P~TELET DEPO~TION BY APO/t-PA
3 Number of Pl Itelets Deposited on Graft Per Microliter Platelets x 10-9 % Inhibition*
Animal # Platelet Count x 10-3 Control Drug Uncorrected=I, Corrected=12 4 Low Oose APC Control Drug 30' 6Q' 30' 60' 30' 60' 30' 60' 87~5 445 2259.3 -- 3.1 S.8 67 -- 34 --86-02 415 445 10.0 13.1 5.2 6.4 48 Sl 52 54 86-00 388 3376.0 -- 3.0 6.2 SO -- 42 --6 M e a n: 51 54 Hi gh Dose APC
7 86-11 173 135 1.5 2.3 0.3 0.5 80 78 74 72 8 B7-15 429 340 14.6 22.7 4.2 3 0 770 87 64 83 9 ean. 83 78 APC Pl us t-PA
87-15 429 38014.6 22.7 6.1 6.7 58 71 53 67 0 jl 87-02 491 430 12.7 ~~ 7.1 __ 51 41 Me~tn: 71 67 11 l 13 l ~ ~) x lOû, Iz = (1 - PDA x PCr) x 100 PD = Platelet Depos;tion A = APC
16 PC = Platelet Count C = Control .

~' I

Claims

1. Use of plasma-derived activated protein C or analogs thereof alone or in combination with a throm-bolytic agent or combinations of thrombolytic agents for inhibiting acute arterial thrombotic occlusion, thromboembolism, or stenosis in coronary, cerebral or peripheral arteries or in vascular grafts.

2. Use of plasma-derived activated protein C or analogs thereof alone or in combination with a throm-bolytic agent or combinations of thrombolytic agents for the manufacture of a medicament for inhibiting acute arterial thrombotic occlusion, thromboembolism, or stenosis is coronary, cerebral or peripheral arter-ies or in vascular grafts.

3. Plasma-derived activated protein C or analogs thereof alone or in combination with a thrombolytic agent or combinations of thrombolytic agents for inhibiting acute arterial thrombotic occlusion, throm-boembolism, or stenosis in coronary, cerebral or peripheral arteries or in vascular grafts.

4. Use according to claim 1 or 2, wherein said thrombolytic agent is tissue plasminogen activator or analogs thereof.

5. Use according to claim 1 or 2, wherein said thrombolytic agent is urokinase or analogs thereof.

6. Use according to claim 1 or 2, wherein said thrombolytic agent is prourokinase or analogs thereof.

7. Use according to claim 1 or 2, wherein said thrombolytic agent is streptokinase or analogs thereof.

8. Use according to claim 1 or 2, wherein said thrombolytic agent is an acylated form of plasminogen or plasmin or analogs thereof.

9. Use according to claim 1 or 2, wherein said thrombolytic agent is acylated streptokinase-plasmino-gen complex or analogs thereof.

10. Use of recombinant produced activated protein C
or analogs thereof alone or in combination with a thrombolytic agent or combinations of thrombolytic agents for inhibiting acute arterial thrombotic occlu-sion, thromboembolism, or stenosis in coronary, cere-bral or peripheral arteries or in vascular grafts.

11. Use of recombinant produced activated protein C
or analogs thereof alone or in combination with a thrombolytic agent or combinations of thrombolytic agents for the manufacture of a medicament for inhibiting acute arterial thrombotic occlusion, throm-boembolism, or stenosis in coronary, cerebral or peripheral arteries or in vascular grafts.

12. Use according to claim 10 or 11, wherein said thrombolytic agent is tissue plasminogen activator or analogs thereof.

13. Use according to claim 10 or 11, wherein said thrombolytic agent is urokinase or analogs thereof.

14. Use according to claim 10 or 11, wherein said thrombolytic agent is prourokinase or analogs thereof.

15. Use according to claim 10 or 11, wherein said thrombolytic agent is streptokinase or analogs thereof.

16. Use according to claim 10 or 11, wherein said thrombolytic agent is an acylated form of plasminogen or plasmin or analogs thereof.

17. Use according to claim 10 or 11, wherein said thrombolytic agent is acylated streptokinase-plasmino-gen complex or analogs thereof.

18. A pharmaceutical composition for inhibiting acute arterial thrombotic occlusion, thromboembolism, or stenosis in coronary, cerebral or peripheral arter-ies or in vascular grafts, the composition comprising an effective amount of activated protein C in a phar-maceutically acceptable carrier.

19. A composition as claimed in claim 18, wherein the activated protein C is selected from the group consisting of plasma-derived activated protein C and recombinantly-derived activated protein C.

20. A composition as claimed in claim 18, wherein the composition further comprises an effective amount of a thrombolytic agent or combination of thrombolytic agents.

21. A composition as claimed in claim 20, wherein the thrombolytic agent is selected from the group con-sisting of tissue plasminogen activator, urokinase, prourokinase, streptokinase, an acylated form of plas-minogen, plasmin, and acylated streptokinase-plasmino-gen complex.

22. A pharmaceutical composition for inhibiting acute arterial thrombotic occlusion, thromboembolism, or stenosis in coronary, cerebral or peripheral arter-ies or in vascular grafts, comprising activated pro-tein C in an amount that provides a dose of about 0.2mg/kg-hr. to 1.1 mg/kg-hr. of activated protein C
in a pharmaceutically acceptable carrier.

23. A composition as claimed in claim 22, wherein the activated protein C is selected from the group consisting of plasma-derived activated protein C or recombinantly-derived activated protein C.

24. A composition as claimed in claim 22, wherein the composition further comprises an effective amount of a thrombolytic agent or combination of thrombolytic agents.

25. A composition as claimed in claim 24, wherein the thrombolytic agent is selected from the group con-sisting of tissue plasminogen activator, urokinase, prourokinase, streptokinase, acylated form of plas-minogen, acylated form of plasmin, and acylated strep-tokinase-plasminogen complex.

26. A composition as claimed in claim 24, wherein the thrombolytic agent is tissue plasminogen activator comprising an amount that provides a dose of about 0.1 to 0.4mg/kg-hr.

27. A pharmaceutical composition for inhibiting acute arterial thrombotic occlusion, thromboembolism, or stenosis in coronary, cerebral or peripheral arter-ies or in vascular grafts, comprising an amount of activated protein C in a pharmaceutically acceptable carrier that provides an activated protein C plasma level in the range of about 0.1 to 1.6µg/ml.

28. A composition as claimed in claim 27, wherein the activated protein C is selected from the group consisting of plasma-derived activated protein C and recombinantly-derived activated protein C.

29. A composition as claimed in claim 27, wherein the composition further comprises an effective amount of a thrombolytic agent or combination of thrombolytic agents.

30. A composition as claimed in claim 29, wherein the thrombolytic agent is selected from the group con-sisting of tissue plasminogen activator, urokinase, prourokinase, streptokinase, acylated form of plas-minogen, acylated form of plasmin, and acylated strep-tokinase-plasminogen complex.

31. A composition as claimed in claim 30, wherein the thrombolytic agent is tissue plasminogen activator comprising an amount that provides a dose of about 0.1 to 0.4mg/kg-hr.

32. A pharmaceutical composition for inhibiting arterial platelet deposition comprising activated pro-tein C in an amount that provides a dose of about 0.2mg/kg-hr. to 1.1mg/kg-hr. of activated protein C in a pharmaceutically acceptable carrier.

33. A composition as claimed in claim 32, wherein the activated protein C is selected from the group consisting of plasma-derived activated protein C and recombinantly-derived activated protein C.

34. A composition as claimed in claim 32, wherein the composition further comprises an effective amount of a thrombolytic agent or combination of thrombolytic agents.

35. A composition as claimed in claim 34, wherein the thrombolytic agent is selected from the group con-sisting of tissue plasminogen activator, urokinase, prourokinase, streptokinase, acylated form of plas-minogen, acylated form of plasmin, and acylated strep-tokinase-plasminogen complex.

36. A pharmaceutical composition for inhibiting arterial platelet deposition comprising an amount of activated protein C in a pharmaceutically acceptable carrier that provides an activated protein C plasma level in the range of about 0.1 to 1.6µg/ml.

37. A composition as claimed in claim 36, wherein the activated protein C is selected from the group consisting of plasma-derived activated protein C and recombinantly-derived activated protein C.

38. A composition as claimed in claim 36, wherein the composition further comprises an effective amount of a thrombolytic agent or combination of thrombolytic agents.

39. A composition as claimed in claim 38, wherein the thrombolytic agent is selected from the group con-sisting of tissue plasminogen activator, urokinase, prourokinase, streptokinase, acylated form of plas-minogen, acylated form of plasmin, and acylated strep-tokinase-plasminogen complex.

40. A composition as claimed in claim 38, wherein the thrombolytic agent is tissue plasminogen activator comprising an amount that provides a dose of about 0.1 to 0.4 mg/kg-hr.